1. Field of the Invention
The present invention relates to detecting the presence of a microorganism in a sample by detecting the presence of its specific DNA sequence using a combination of complementary DNA sequences with one linked to a fibrinogen-splitting enzyme-based bio-chemo-physical conversion method and an ultrasonic microorganism detection apparatus.
2. Description of the Related Art
A variety of methods exists in the detection of the presence of a biological analyte, for examples, enzyme immunoassay (EIA), radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR), real-time-PCR (RT-PCR), etc. These methods have been used to measure levels of hormones, antigens, antibodies, enzymes, proteins, drugs, specific stretches, etc. of DNAs and pollutants.
Among these methods, the most popular ones are PCR and RT-PCR in which samples containing microorganisms of interest will have their DNAs extracted. Then, the genomic double strand DNAs will be heated to 94-96° C. and held for 1-9 minutes so that the hydrogen bonds between the complementary bases of the DNA strands break and give single strands of DNA. In order to detect the presence of a microorganism of interest in a sample, complementary DNA oligonucleotide primers (e.g. F#1 and R#1 for forward and reverse sequences, respectively) are added to bind to adjacent stretches of DNA sequences that are either up-stream or down-stream of this specific stretch of DNA (in the single strands of DNA) of interest (sequence #1). Stable DNA-DNA hydrogen bonds will be formed when the primer sequences match very closely the template sequence, and the process is referred to as annealing. The reaction temperature will then be lowered to 50-65° C. before being kept for 20-40 seconds. With the addition of deoxynucleotide triphosphates and through the actions of DNA polymerase (e.g. Taq polymerase), a DNA fragment (called sequence #2) which is complementary to this specific sketch of DNA of interest (sequence #1) and indicative of the presence of microbes of interest will be manufactured. Sequence #1 is said to be transcribed and sequence #2 is formed. This is called the extension/elongation step. With repeated cycles of warming up (90° C.) and cooling down (50 to 65° C.), sequence #1 will be transcribed many times (usually 30-35 times) before the final elongation step (70 to 74° C. for 15 minutes). This ensures that any remaining single-stranded DNA is fully extended. With the PCR method, the mixture containing the transcribed stretches of specific DNA (i.e. sequence #2, the primers, the enzymes, etc.) will have to be resolved with agarose gel electrophoresis using 1% agarose gels. These DNA fragments and primers will be resolved by their molecular weights. Visualization of the DNA fragments is achieved by the addition of ethidium bromide under ultra-violet (UV) lights. If the microorganism of interest is present in the sample, after the PCR steps, an UV-absorbing DNA band of the right molecular size as the complementary DNA (sequence #2) will be present. Subsequently, this PCR product (supposedly sequence #2) has to be inserted in a bacteriophage vector system (e.g. pGEM-T) before being expressed in a host bacterium (e.g. E. Coli). DNA sequence of this DNA band (i.e. sequence #2) can then be investigated using traditional dideoxy methods. If and when sequencing results obtained for this PCR product (experimentally obtained sequence #2) is complementary to that of sequence #1, the test will be considered affirmative. On the other hand, with the RT-PCR method, DNA binding dyes (such as Cyanine green dyes) will be added to the reaction mixture (with all the other reagents) in the initial heating step. With similar experimental conditions to those of the PCR method, if the specific sketch of DNA (sequence #1) is present in the sample, it will be transcribed by the DNA polymerase presented. Many copies of sequence #2 will be produced with repeatedly transcribed. As the amount of DNA sequences increases and with the presence of DNA binding dyes, the amount of dyes bound to the DNA will be increased. This dye has the unique fluorescent characteristics and will fluoresce when bound to DNA. Therefore, an increased florescence not only indicates the presence of this complementary DNA sequence (sequence #2) that is indicative of the presence of this microorganism sequence of interest (sequence #1) and hence the microorganism of interest, but also indicates the number of copies of this sketch of DNA (sequence #1) in the microorganism of interest.
Nonetheless, these PCR and RT-PCR methods, while capable of being used on-site for field operations, they involve relatively heavy instrumentation as they rely on fluorescence to detect and quantify levels of the analyte presented. Hence, these methods with relatively heavy instrumentation are not suitable for on-site field operations.
The present invention does not require a florescence source to detect the presence of a microorganism of interest. In addition, each component shown in FIG. 1 can be only a few centimeters large. As such, the size of the microorganism detection apparatus can be relatively small (e.g. 18 cm long, 8 cm wide and 5 cm tall as in our prototype). Furthermore, the microorganism detection apparatus can be wholly battery operated. Thus, the apparatus can be operated at a remote location without regular electrical power supply. Due to its light-weight, portability and the simplicity of operation, the apparatus is suitable for handheld on-site field operations. Through the present invention, the disadvantages of prior detection methods and apparatuses are overcome and benefits are realized in the field of biological analyte analysis.